Blood coagulation tests may be performed for a variety of purposes, including determining the bleeding susceptibility of patients undergoing surgery, and monitoring patients undergoing anti-coagulation therapy for the prevention of blood clots. A variety of coagulation tests are presently in use, for example, the Activated Partial Thromboplastin Test (APTT), and Prothrombin Time (PT) test. Both tests measure clotting time to evaluate a patient's baseline hemostatic state or to monitor the response to anticoagulant therapy.
The APTT test is used for the evaluation of the intrinsic and common coagulation pathways, and for monitoring therapy with unfractionated heparin and other anticoagulants. The APTT measures the time in seconds required for a fibrin clot to form in a plasma sample to be tested after a partial thromboplastin reagent (an activating agent) and calcium chloride have been added to the sample. The APTT test is widely used for monitoring heparin therapy.
The PT test relies upon the induction of the extrinsic coagulation protease factor VIIa by thromboplastin in a blood sample to be tested. The extrinsic coagulation pathway results in the production of thrombin, a proteolytic enzyme that catalyzes the conversion of fibrinogen to fibrin, which is essential to the clotting process. The PT test utilizes this series of enzymatic events in vitro under controlled conditions to diagnose dysfunctions or deficiencies in the blood coagulation system of patients. The PT test is also used in determining a safe and effective dose of an anticoagulant. The amount of time (in seconds) that elapses until clot formation occurs is the Prothrombin Time, or PT value.
Anticoagulant therapy for acute thromboembolic disease typically consists of parenteral heparin followed by oral warfarin. The pharmacodynamic properties of warfarin necessitate a period of overlapping therapy with heparin until a steady-state warfarin anticoagulant effect is achieved. Heparin (unfractionated) is a polysaccharide with sulfate groups attached by covalent bonding. Heparin is heterogenous in terms of molecular weight and the degree of sulfation. The molecular weight ranges between about 5,000 to about 30,000 daltons. The anti-coagulant effect ofheparin in blood results from the binding and activation of a plasma protein, antithrombin III (AT III), which inhibits enzymes in the coagulation cascade. In particular, the heparin-AT III complex inhibits the coagulation activity of Factors Xa and IIa (thrombin). R. D. Rosengerg et al., Hemostasis and Thrombosis, ed. R. Colman et al., p. 711 (2001). An infusion of excess heparin in a patient, however, may cause bleeding problems.
The administration of heparin is typically monitored using the Activated Partial Thromboplastin Time (APTT) assay. The APTT assay generally consists of three steps: 1) addition of an activator to plasma or blood; 2) incubation of two to four minutes; and, 3) addition of calcium chloride solution, after which the clotting time is monitored. The APTT assay provides a prolonged clot time in the presence of heparin. As an alternative to the APTT assay, a one-step prothrombin time (PT) assay has been investigated. A coagulation assay system using a PT reagent is a simpler and faster method requiring only one reagent (PT), and does not require incubation time, as opposed to an APTT system for heparin which requires two reagents (activating agent and calcium chloride) and two to five minutes for incubation. J. P. Miletich, Prothrombin Time, Williams Hematology, Fifth Ed., Ed. by E. Beutler, pp. L82-L86 (1995a and 1995b).
With regard to a PT system for assaying heparin, Schultz et al. reported that the clotting time difference between 0.2 and 0.4 units per ml of heparin (therapeutic range) in citrated plasma is only a few seconds. N. J. Schultz, et al., The Influence of Heparin on the Prothrombin Time, Pharmacotherapy, Vol. 11, No. 4, pp 312-316 (1991). The clotting time separation of only a few seconds, however, is too narrow for the measurement of heparin levels in plasma or a blood sample to use a PT reagent.
Low molecular weight heparin (LMWH), as an alternative to standard heparin, is derived from unfractionated heparin through either chemical or enzymatic depolymerization. R. J. Linhardt et al., Semin. Thromb. Hemost. Suppl., 3, pp.5-16 (1999). LMWH has a molecular weight ranging from about 3,000 to about 4,500 daltons. The difference in molecular weight results in properties that are distinct from those of standard or traditional heparin. For example, LMWH binds less strongly to protein, has enhanced bioavailability, and interacts less with platelets. As with standard heparin, LMWH binds to antithrombin III, but inhibits thrombin to a lesser degree (and Factor Xa to a greater degree) than standard heparin. LMWH is increasingly being used in patients with unstable angina, deep vein thrombosis, and percutaneous coronary intervention.
The activity of LMWH may be expressed in terms of anti-Xa activity. The anti-Xa activity of LMWH in patient blood samples is usually determined by chromogenic assay with the isolated plasma. This is a time consuming assay using an expensive procedure. In recent publications, LMWH has been described as having significant anti-IIa activity, L. Bara et al., Thromb. Res., 69,443-452 (1993), as accumulating in patients of renal failure, M. Samama, Thromb. Haemost., 15, 199 (1995), and as causing bleeding problems in some patients involving surgery to the knee and hip, Shaieb, M. D., et al., J. Arthroplasty, 14, 432-438 (1999), and to the spine, Lumpkin, M. M., Int. J. Trauma Nurs., 4, 56-57 (1998). Hence, there is an increasing demand in the medical field for monitoring the effects of LMWH.
Holmes, et al., reported on low molecular weight heparin determination by PT assay, showing a good separation of clotting times between control blood and therapeutic range (0.6 to 1.0 u/ml) of a blood sample treated with Enoxaparin, a low molecular weight heparin preparation available from Aventis Corporation. Enoxaparin is also available, under the trademark LOVENOX®, from Aventis Corporation. In the system described by Holmes, et al., the clotting time for 1.0 u/ml of Enoxaparin is above 400 seconds, and an expensive corn trypsin inhibitor is required due to the lengthy clotting times, i.e., 272 to 486 seconds. M. B. Holmes, et al., Novel, Bedside, Tissue Factor-Dependent Clotting Assay Permits Improved Assessment of Combination Antithrombotic and Antiplatelet Therapy, Circulation, 102, pp.2051-2057 (2000).
A method of monitoring LMWH in a citrated plasma sample by prothrombin time assay was described by Wu in WO 99/10746. In the Wu method, commercially available thromboplastin (PT reagent) was used by diluting 200 to 6,400-fold in buffered calcium chloride solution, followed by mixing with citrated plasma for the clotting assay. The composition and process of preparation of the commercially available thromboplastin, however, were not disclosed. The reagent described by Wu is a liquid state reagent.
An air-dried PT reagent for the assay of plasma prothrombin time is described by Lee et al., in U.S. Pat. No. 6,183,979, assigned to the assignee herein, the entire disclosure of which is hereby incorporated by reference, but the PT reagent described is not suitable for monitoring low molecular weight heparin.
There remains a need for a highly sensitive and stable reagent and assay for measuring the concentration of low molecular weight heparin in blood.